This invention relates to novel nitroso compounds and to their use as spin traps.
It is now widely accepted that Reactive Oxygen (RO) and Reactive Nitrogen (RN) species (including free radicals) are involved in the pathogenesis of several disease states. The only technique which can detect low concentrations of radicals directly is electron spin resonance (ESR) spectroscopy. Although this technique is highly sensitive (thresholds of 10xe2x88x927-10xe2x88x926M spins), it is not directly applicable to the study of biological oxidations. A more useful method, permitting ESR investigation of short-lived reactive free radicals by transforming them into more persistent species, is the so-called xe2x80x9cspin trappingxe2x80x9d method. The technique of spin trapping makes use of a diamagnetic compound in which all the electrons are in pairs. The diamagnetic compound, known as the spin trap, reacts with a free radical (Rxe2x80x2) which contains an unpaired electron with spin. Reaction of the spin trap with a reactive free radical, results in the formation of a relatively stable, ESR-observable spin adduct. In favourable cases, the free radical, Rxe2x80x2, can be identified from the ESR parameters of the spin adduct (e.g. hyperfine coupling constants, g-factor).
This technique was established by Jansen et al, JACS (1968) 90:5909-10. Since then, there has been much research on the synthesis of suitable spin traps. The spin traps that have been most commonly employed are those designed so that, on reaction with a free radical, a nitroxide is formed.
A commercially available spin trap is 3,5-dibromo-4-nitrosobenzenesulphonate, sodium salt (DBNBS). See Kaur et al, JCS Chem. Comm. (1981) 142-3. DBNBS is a water-soluble aromatic C-nitroso spin trap which has been reported to trap the sulphite radical anion (SO3xe2x88x92), superoxide, alkyl, nitric oxide and selenite anion radicals. DBNBS has also been reported to detect an oxidising species in uremic plasma, as it is oxidized to its radical cation DBNBS+ which is then detected by ESR spectroscopy. See Roselaar et al, Kidney International (1995) 48:199-206, and WO-A-92/18874.
Reist et al, FEBS Lett. (1998) 423:231-4, indicates that sulphite is toxic to the lung and can cause allergic reactions such as bronchoconstriction in asthmatics. The toxic effects of sulphite in combination with peroxynitrite neuronal cells are reported by Reist et al, J. Nephrology (1998) 71:2431-8. An effective spin trap for sulphite is therefore of great potential value.
Matsuo et al, Free Radical Biology and Medicine (1998) 25:929-35, reports a very sensitive xe2x80x98ELISA-ESRxe2x80x99 method for Hepatitis B surface (HBs) antigen detection, using 4-hydrazonomethyl-1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide (HHTIO) as the spin trap. In this method, beads are coated with the first HBs-antibody, and a second HBs-antibody is labelled with horseradish peroxidase (HRP). In the presence of HBs antigen, the HRP-labelled antibody will be linked to the antigen and the antigen linked to the beads. After washing, the antigen-antibody complex is added to a solution containing p-acetamidophenol (p-AP), hydrogen peroxide and HHTIO. The p-AP is converted into phenoxy radicals by the action of HRP in the presence of hydrogen peroxide, and the phenoxy radical is trapped by HHTIO to form a stable nitroxide radical which can be detected by ESR spectroscopy.
Novel compounds according to this invention are 3,5-dichloro-4-nitrosobenzenesulphonate (DCNBS) and 3,5-dimethyl-4-nitrosobenzenesulphonate (DMNBS) and salts thereof.
DCNBS at least has several advantages over DBNBS. These include improved solubility of the spin trap in aqueous systems and narrower ESR signals (which result in a greater signal/noise ratio).
DCNBS has been used successfully to detect an oxidant in the dialysate of patients with renal failure (the oxidant oxidized DCNBS to its radical cation DCNBS+ which was subsequently detected by ESR spectroscopy).
DCNBS will trap nitric oxide. It is also a potential spin trap for alkyl free radicals, superoxide and the selenite radical anion (SeO3xe2x88x92).
DCNBS has been found to be a more sensitive spin trap than DBNBS for the sulphite radical anion (SO3xe2x88x92). DMNBS is also superior to DBNBS or its isotopic analogues (15N and d2), or DCNBS. DMNBS was shown to give an ESR signal for the SO3xe2x88x92 adduct of more than 20 times that obtained with DBNBS.
Accordingly, DMNBS as a spin trap for the sulphite radical anion has enormous potential. In addition, DMNBS may be used as a detector molecule for the presence of antibody-peroxidase complexes in ELISAs with ESR detection.